Biotechnology 3 Unit 10 ReviewBiochemistry in Biotech

Key Concepts and Fundamentals

• Biochemistry studies chemical processes within living organisms focuses on structure, function, and interactions of biological molecules
• Involves understanding the properties of water essential for life due to its polarity, hydrogen bonding, and ability to dissolve polar and ionic compounds
• Includes the study of pH and buffers critical for maintaining stable cellular environments and optimal conditions for biochemical reactions
• Covers the structure and function of the four major classes of biomolecules: carbohydrates, lipids, proteins, and nucleic acids
• Explores the concept of thermodynamics and energy flow in biological systems, including the role of ATP as the primary energy currency
• Examines the principles of enzyme catalysis and how enzymes lower activation energy and speed up biochemical reactions
• Investigates the central dogma of molecular biology, which describes the flow of genetic information from DNA to RNA to proteins

Biomolecules and Their Functions

• Carbohydrates serve as energy storage molecules (glycogen), structural components (cellulose), and cell signaling molecules (glycoproteins)
  • Monosaccharides are simple sugars (glucose, fructose) that form the building blocks of more complex carbohydrates
  • Disaccharides consist of two monosaccharides joined by a glycosidic bond (sucrose, lactose)
  • Polysaccharides are long chains of monosaccharides (starch, chitin)
• Lipids are hydrophobic molecules that include fats, oils, waxes, and steroids play crucial roles in energy storage, cell membrane structure, and signaling
  • Triglycerides are the primary form of energy storage in animals consist of three fatty acids attached to a glycerol backbone
  • Phospholipids are the main components of cell membranes have a hydrophilic head and two hydrophobic tails
• Proteins are polymers of amino acids that perform a wide range of functions, including catalysis (enzymes), transport (hemoglobin), structural support (collagen), and immune defense (antibodies)
  • The primary structure of a protein is the linear sequence of amino acids
  • Secondary structure refers to local folding patterns (α-helices and β-sheets) stabilized by hydrogen bonds
  • Tertiary structure is the overall 3D shape of a protein determined by interactions between amino acid side chains
  • Quaternary structure involves the assembly of multiple protein subunits into a functional complex
• Nucleic acids, DNA and RNA, store and transmit genetic information
  • DNA is a double-stranded helix composed of nucleotides containing deoxyribose sugar, phosphate, and one of four nitrogenous bases (A, T, C, G)
  • RNA is single-stranded composed of nucleotides with ribose sugar and uracil (U) instead of thymine (T)

Metabolic Pathways and Energy Production

• Metabolism encompasses all chemical reactions in cells divided into catabolic (breaking down molecules) and anabolic (building molecules) processes
• Glycolysis is a central metabolic pathway that breaks down glucose into pyruvate, producing ATP and NADH
  • Occurs in the cytoplasm does not require oxygen
  • Key enzymes include hexokinase, phosphofructokinase, and pyruvate kinase
• Citric acid cycle (Krebs cycle) is a series of reactions that oxidize acetyl-CoA to CO2, generating NADH, FADH2, and ATP
  • Takes place in the mitochondrial matrix
  • Key enzymes include citrate synthase, isocitrate dehydrogenase, and α-ketoglutarate dehydrogenase
• Oxidative phosphorylation is the process by which cells use the electron transport chain to create a proton gradient and drive ATP synthesis
  • Electrons from NADH and FADH2 are transferred through a series of protein complexes (I, II, III, IV) in the inner mitochondrial membrane
  • Proton gradient is used by ATP synthase to generate ATP
• Photosynthesis is the process by which plants and other autotrophs convert light energy into chemical energy stored in glucose
  • Light-dependent reactions occur in the thylakoid membranes of chloroplasts and involve photosystems I and II, which capture light energy to generate ATP and NADPH
  • Light-independent reactions (Calvin cycle) take place in the stroma and use ATP and NADPH to fix CO2 into glucose

Enzyme Kinetics and Regulation

• Enzymes are biological catalysts that speed up chemical reactions by lowering the activation energy
  • Active site is the region of an enzyme where the substrate binds and the reaction occurs
  • Enzymes are highly specific to their substrates due to the shape and chemical properties of the active site
• Michaelis-Menten kinetics describes the relationship between substrate concentration and reaction rate for many enzymes
  • $V_0 = \frac{V_{max}[S]}{K_m + [S]}$ where $V_0$ is the initial reaction rate, $V_{max}$ is the maximum rate, $[S]$ is substrate concentration, and $K_m$ is the Michaelis constant
  • $K_m$ is the substrate concentration at which the reaction rate is half of $V_{max}$ and indicates the affinity of the enzyme for the substrate
• Enzyme activity can be regulated through various mechanisms
  • Competitive inhibition occurs when a molecule similar to the substrate binds to the active site, preventing substrate binding
  • Non-competitive inhibition involves an inhibitor binding to a site other than the active site, altering the enzyme's conformation and reducing its activity
  • Allosteric regulation involves the binding of effector molecules at sites distant from the active site, causing conformational changes that affect enzyme activity
  • Covalent modification, such as phosphorylation or acetylation, can alter enzyme activity by changing its conformation or binding properties
• Enzyme activity is also influenced by environmental factors such as temperature, pH, and ionic strength
  • Optimal temperature and pH vary among enzymes and reflect the conditions in which they function best
  • Deviations from optimal conditions can lead to enzyme denaturation and loss of activity

Nucleic Acids and Protein Synthesis

• DNA replication is the process by which cells duplicate their genetic material before cell division
  • Semiconservative replication means each new DNA molecule contains one original strand and one newly synthesized strand
  • DNA polymerases catalyze the addition of nucleotides to the growing strand, using the original strand as a template
  • Replication begins at specific sites called origins of replication and proceeds bidirectionally
• Transcription is the synthesis of RNA from a DNA template, carried out by RNA polymerases
  • In prokaryotes, transcription occurs in the cytoplasm and is performed by a single RNA polymerase
  • In eukaryotes, transcription takes place in the nucleus and involves three types of RNA polymerases (I, II, and III)
  • Transcription factors bind to specific DNA sequences (promoters and enhancers) to regulate gene expression
• Translation is the process of synthesizing proteins from mRNA templates, carried out by ribosomes
  • Ribosomes consist of two subunits (large and small) composed of ribosomal RNA (rRNA) and proteins
  • tRNAs (transfer RNAs) act as adaptor molecules, carrying specific amino acids to the ribosome and recognizing codons in the mRNA
  • The genetic code is the set of rules that determines which amino acid corresponds to each three-nucleotide codon
  • Translation involves three main stages: initiation (assembly of the ribosome on the mRNA), elongation (addition of amino acids to the growing polypeptide chain), and termination (release of the completed protein)
• Post-translational modifications, such as glycosylation, phosphorylation, and proteolytic cleavage, can alter the structure, function, and localization of proteins

Biotechnology Applications

• Recombinant DNA technology involves the manipulation and insertion of genes from one organism into another
  • Restriction enzymes are used to cut DNA at specific sequences, allowing the isolation and manipulation of genes
  • DNA ligase is used to join DNA fragments, enabling the creation of recombinant DNA molecules
  • Plasmids and viral vectors are commonly used to introduce recombinant DNA into host cells
• Polymerase chain reaction (PCR) is a technique used to amplify specific DNA sequences
  • Involves repeated cycles of denaturation, primer annealing, and extension using a heat-stable DNA polymerase (Taq polymerase)
  • Enables the rapid generation of large quantities of a desired DNA fragment for analysis or cloning
• Genetically modified organisms (GMOs) are organisms whose genetic material has been altered using genetic engineering techniques
  • Agricultural applications include the development of crops with increased yield, resistance to pests and herbicides, and improved nutritional content (Golden Rice)
  • Medical applications include the production of recombinant proteins (insulin) and the development of gene therapies for genetic disorders (sickle cell anemia)
• Biopharmaceuticals are drugs produced using living organisms or their components
  • Monoclonal antibodies are highly specific antibodies produced by identical immune cells, used for targeted therapies (Herceptin for breast cancer)
  • Recombinant proteins, such as human growth hormone and erythropoietin, are produced in genetically engineered cells and used to treat various conditions
• Bioremediation involves the use of microorganisms or their enzymes to degrade or neutralize pollutants in the environment
  • Bacteria capable of breaking down oil (Alcanivorax borkumensis) have been used to clean up oil spills
  • Genetically engineered plants (poplar trees) have been developed to absorb and accumulate heavy metals from contaminated soils

Lab Techniques and Experiments

• Gel electrophoresis is a technique used to separate DNA, RNA, or proteins based on their size and charge
  • Agarose gel electrophoresis is commonly used for DNA and RNA, while polyacrylamide gel electrophoresis (PAGE) is used for proteins
  • Molecules are separated by applying an electric field to a gel matrix, with smaller molecules migrating faster than larger ones
• Chromatography is a family of techniques used to separate mixtures based on the differential partitioning of components between a stationary phase and a mobile phase
  • Affinity chromatography uses a stationary phase with specific ligands to selectively bind and purify target molecules (His-tagged proteins)
  • Size-exclusion chromatography separates molecules based on their size, with larger molecules eluting earlier than smaller ones
• Spectrophotometry is the measurement of the absorption or transmission of light by a sample
  • UV-Vis spectrophotometry measures the absorption of ultraviolet and visible light and is used to quantify the concentration of biomolecules (DNA, proteins)
  • Spectrophotometric enzyme assays monitor the change in absorbance of a substrate or product to measure enzyme activity
• Centrifugation is a technique that uses centrifugal force to separate particles based on their size, shape, and density
  • Differential centrifugation involves a series of centrifugation steps at increasing speeds to separate cellular components (nuclei, mitochondria, cytosol)
  • Density gradient centrifugation uses a gradient of increasing density to separate molecules or organelles based on their buoyant density
• Microscopy is the use of microscopes to visualize objects too small to be seen with the naked eye
  • Light microscopy uses visible light and a system of lenses to magnify samples, with a resolution limit of about 200 nm
  • Electron microscopy (scanning and transmission) uses a beam of electrons to create high-resolution images, with a resolution limit of about 0.1 nm
  • Fluorescence microscopy uses fluorescent dyes or proteins (GFP) to label specific molecules or structures within cells, allowing their visualization and localization

Current Research and Future Directions

• Single-cell genomics and transcriptomics aim to study the genetic material and gene expression patterns of individual cells
  • Enables the identification of rare cell types and the understanding of cellular heterogeneity within tissues
  • Helps uncover the molecular mechanisms underlying complex biological processes (development, disease progression)
• CRISPR-Cas9 is a powerful genome editing tool that allows precise modification of DNA sequences
  • Consists of a guide RNA that directs the Cas9 endonuclease to a specific DNA sequence, where it creates a double-strand break
  • Can be used to introduce targeted mutations, correct genetic defects, or regulate gene expression
  • Potential applications include the development of disease-resistant crops, the treatment of genetic disorders, and the creation of animal models for research
• Synthetic biology is an interdisciplinary field that aims to design and construct novel biological systems or organisms with desired functions
  • Involves the use of standardized genetic parts (BioBricks) and the application of engineering principles to biology
  • Examples include the creation of synthetic metabolic pathways for the production of biofuels or pharmaceuticals and the development of biosensors for environmental monitoring
• Personalized medicine aims to tailor medical treatments to an individual's genetic profile, lifestyle, and environment
  • Pharmacogenomics studies how genetic variations influence drug response and helps optimize drug selection and dosage for individual patients
  • Targeted therapies, such as small molecule inhibitors and monoclonal antibodies, are designed to specifically target molecular pathways or proteins involved in disease
• Microbiome research investigates the diverse microbial communities that inhabit the human body and their role in health and disease
  • The human gut microbiome has been linked to various conditions, including obesity, inflammatory bowel disease, and neurological disorders
  • Probiotics and fecal microbiota transplantation are being explored as potential therapies for microbiome-related diseases
• Bioinformatics and computational biology are increasingly important for managing and analyzing the vast amounts of biological data generated by high-throughput technologies
  • Genome sequencing projects have generated massive datasets that require sophisticated computational tools for assembly, annotation, and comparison
  • Machine learning and artificial intelligence are being applied to biological data to identify patterns, predict protein structures, and discover new drug targets